Unlike other restriction enzymes, Bfi I functions without metal ions. It recognizes an asymmetric DNA sequence, 5′-ACTGGG-3′, and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like Fok I, dimerize transiently during catalysis. Bfi I is also a dimer but it has only one active site, at the dimer interface. We show here that Bfi I remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and/or the DNA in the Bfi I–DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting Bfi I into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5′ phosphate in the bottom strand. Bfi I thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.
Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains.
1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme–substrate complex.
Abstract An alkaline phosphatase-negative (P-) mutant of Escherichia coli K-12 that produces a protein antigenically related to the wild type enzyme has been found to accumulate a zinc-deficient dimeric form of this metalloenzyme. The mutationally altered alkaline phosphatase could be activated by incubation at pH values between 5 and 6 in the presence of Zn(II) at 37°. The kinetics of the activation process was examined with purified preparations of the apoenzyme. Activation appeared to consist of multiple stages of structural rearrangements induced by zinc binding. The pH sensitivity of the activation of the isolated protein could be correlated with a pH conditionality of the P- phenotype. Thus, the mutant acted as a P- strain when grown at pH 7.4, but was P+ when grown at pH 6.0. Although unable to grow effectively in a medium containing an organic phosphate as the sole phosphate source at pH 7.4, the P- strain survived much better than its P+ parent in a medium totally lacking phosphorus.
Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5' terminus of the cleaved DNA. Under certain conditions, the terminal 3'-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.
Proteins that bind to specific sequences in long DNA molecules have to locate their target sites amid myriad alternative sequences, yet they do so at remarkably rapid rates, sometimes approaching 1010 M−1·s−1. Hence, it has been asserted widely that binding to specific DNA sites can surpass the maximal rate for 3D (three-dimensional) diffusion through solution and that this could only be accounted for by a reduction in the dimensionality of the search for the target in effect by 1D (one-dimensional) diffusion (or ‘sliding’) along the DNA contour. It will be shown here that there is, in fact, no known example of a protein binding to a specific DNA site at a rate above the diffusion limit, and that the rapidity of these reactions is due primarily to electrostatic interactions between oppositely charged molecules. It will also be shown that, contrary to popular belief, reduced dimensionality does not, in general, increase the rate of target-site location but instead reduces it. Finally, it will be demonstrated that proteins locate their target sites primarily by multiple dissociation/reassociation events to other (nearby or distant) sites within the same DNA molecule, and that 1D diffusion is limited to local searches covering ∼50 bp around each landing site.
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